1. Field Of The Invention
The present invention concerns the field of molecular biology and more particularly HMGB1protein inhibitors and HMGB1 antagonists to be used for the treatment of vascular diseases, including those due to angioplasty.
2. Description Of The Related Art
HMGB1 protein(known, before 2001, as HMG; Bustin, 2001, Trends Biochem. Sci., 26, 152-153) is the archetypal protein of the HMG-box family, which is characterised by the presence of DNA binding domains, called HMG boxes. HMG1 is a small 25-kD protein, of 215 amino acids, with a highly conserved sequence among mammals. The HMGB1 molecule is organised into three domains: two DNA binding domains, HMG Box A and BoxB, which are followed by an acidic COOH terminus composed of 30 glutamic and aspartic residues. The two HMG boxes, box A and boxB, are 80 amino acid segments(29% identical, 65% similar), having an L-shaped tridimensional structure (Hardman et al., 1995, Biochemistry, 34 :16596-16607 Read et al., 1993, Nucleic Acids Res., 21: 3427-3436; Weir et al., 1993, EMBO J., 12: 1311-1319).
HMGB1 has originally been identified as a ubiquitously expressed, abundant nuclear protein. It is present in more than 1 million copies per single nucleus and binds double stranded DNA without sequence specificity.
Instead, HMGB1 binds with high affinity to specific DNA structures like kinked or bent DNA and four-way junctions. However, HMGB1 can be recruited to double stranded DNA by interaction with several different DNA binding proteins. When bound to double stranded DNA, it induces structure distortion, allowing the formation of nucleoprotein complexes where several DNA-binding proteins can contact each other while bound to their respective DNA cognate sites (Muller et al., 2001, EMBO J., 16: 4337-4340 and other reference cited herewithin). The phenotype of HMGB1-/-mice is in agreement with this mode I(Calogero et al., 1999, Nat. Genet., 22: 276-280).
Recently, an additional role for HMGB1 outside the cell nucleus has come into focus: HMGB1 works as late mediator of endotoxin-induced lethality as well as acute lung inflammation in mice; as well the elevated serum level of HMGB1 in septic patients is a prognosis marker (international patent application No. WO 00/47104). HMGB1 can be secreted by macrophages and pituicytes in culture in response to cytokines and bacterial endotoxin (Abraham et al., 2000, J. Immunol., 165:2950-2954; Wang et al., 1999, Surgery (St. Luis), 126 :389-392; Wang et al., 1999, Science, 285 : 248-251).
The release of HMGB1 from murine erythroleukemia cells is correlated with cell differentiation and the protein can be found in a plasma membrane-associated form in these cells (Passalacqua et al., 1997, FEBS Lett., 400 : 275-279; Sparatore et al. 1996, Biochem. J., 320: 253-256). A protein called amphoterin, identical in sequence to HMGB1, has been described in the brain, where it is found in the nucleus and cytoplasm of neuronal cells as well as in the extracellular space.
If exogenously added, HMGB1 mediates outgrowth of neurites, and laminin-dependent migration of neuroblastoma and glioma cells is inhibited by antibodies against HMGB1 (Fages et al., 2000, J. Cell Sci., 113: 611-620; Merenmies et al., 1991, J. Biol. Chem., 266 : 16722-16729; Parkkinen et al., 1993, J. Biol. Chem., 268: 19726: 19738; Rauvala et al., 1988, J. Cell Biol., 107: 2293-2305). Interactions between HMGB1 and the plasminogen activation system, in particular t-PA (tissue-type plasminogen activator), results in enhanced plasmin formation (Parkkinen and Rauvala, 1991, J. Biol. Chem., 266: 16730-16735). Degradation of extracellular matrix proteins is an important step in the cell migration process, and HMGB1-promoted increase of extracellular protease activity might enable the cells to migrate.
HMGB1 has been identified as one of the ligands binding to the RAGE receptor (Receptor for advanced glycation endproducts)(Hori et al., 1995, J. Biol. Chem., 270:25752-25761). RAGE is a multiligand receptor of the immunoglobulin super family and is expressed in many cell types, including endothelial cells, smooth muscle cells, mononuclear phagocytes, and neurons (Brett et al., 1993, Am. J. Phathol., 143 : 1699-1712; Neeper et al., 1992, J. Biol. Chem., 267: 14998-15004). It is implicated in several different pathological processes, such as diabetes, amyloidoses, and atherosclerosis (Schmidt et al., 1999, Circ. Res., 84: 489-497). Interaction of HMGB1 and RAGE induces neurite outgrowth, and the two proteins colocalize at the leading edge of advancing neurites during embryonic development (Huttunen et al., 1999, J. Biol. Chem., 274:19919-19924). The block of tumour growth and metastasis is observed preventing the interactions between HMGB1 and RAGE; moreover, inhibition of this interaction suppresses activation of mitogen-activated protein (MAP) kinases and the expression of matrix metalloproteinases, molecules importantly linked to tumour proliferation and invasion (Taguchi et al., 2000, Nature, 405: 354-360).
The inventors of the present invention, demonstrated that HMGB1 has a potent biological effect on smooth muscle cells (SMC), one of the cell types where RAGE is expressed on the surface. Vascular SMC cells are the most predominant cells of the larger blood vessels; they are located in the tunica media where are embedded in the extracellular matrix. In intact vessels, SMC cells are in a contractile state and show a phenotype characterised by the absence of cell division and migration responsible for vessel wall rigidity and elasticity maintenance and blood pressure control.
When the endothelium is damaged, either after mechanical or inflammatory injuries, SMC cells switch to a synthetic phenotype and undergo cell division and cell migration. The migration of SMC cells from the tunica media to the tunica intima, resulting in intimal thickening, plays an important role in the pathophysiology of many vascular disorders, such as atherosclerosis and restenosis after coronary angioplasty. In the synthetic state, SMC cells also produce higher amounts of extracellular proteinases, growth factors, and cytokines and secrete a fibrous extracellular matrix. After vessel wall injury, the release of several growth factors and/or chemoattractants either by circulating monocytes, macrophages and platelets, or by damaged endothelial cells can induce SMC cells switch from the contractile to the synthetic phenotypes and it can direct the migration of SMC cells towards the vessel intima. Among these factors, bFGF appears to be one of the most important, but however, SMC cells can also start migration in response to angiogenic stimuli (Schwartz, 1997, J. Clin. Invest., 99: 2814-2816; Van Leeuwen, 1996, Fibrinolysis, 10:59-74).